Description

The MazeEngineers Place preference chamber is created to allow for maximum flexibility for your experiments. The chamber comes with 2 divider slots that allow the zebrafish to choose a chamber or retain the zebrafish in the center area. Floor is clear to allow for location preference. Customized floor patterns can be included in your order upon request. Sizing modifications can be requested within 50% of the original sizing. Please inquire for more details

Frequently Asked Questions

1. Do you provide floor patterns?
   • Due to the wide variety of floor patterns, we do not provide these. Typically, the apparatus is placed on top of a printed sheet of plastic covered paper to allow for waterproof wide ranging patterns at low cost.
2. Can you provide floor patterns?
   • Yes, we are happy to provide floor patterns, custom made on request. Please inquire for more details.
3. Are the Dividers removable?
   • yes the dividers can be removed and reslotted. We provide 4 inserts, 2 with hole patterns and 2 without so that you can retain the zebrafish in the middle area or allow the zebrafish choice preference.

Documentation

Introduction

Zebrafish conditioned place preference (CPP) is a widely used paradigm to evaluate the rewarding effects of food substances or psychoactive Compounds. In the CPP task, the positive-reinforcing effects of substances are assessed by the preference of the zebrafish to linger in the environment that was previously conditioned with a substance.

The CPP paradigm exploits the natural tendency of the zebrafish to choose black over white. In this assay, the primary stimulus serves as an unconditioned stimulus. When it is paired with a secondary stimulus (visual cue) which acts as a conditioned stimulus, an approaching behavior for the paired environment is evoked. (Darland & Dowling, 2001)

The CPP procedure is generally segregated into three testing phases. During Phase I, the subjects are allowed to explore all the compartments of CPP tank freely. The time spent in each compartment is recorded as the baseline place preference. During Phase II, the subjects are confined to each compartment receiving either experimental or control treatment for a specific period of time. In Phase III, the final preference is recorded by permitting the subjects to move freely in all compartments once again. The time spent in each compartment is recorded as the final place preference.  CPP behavior is quantitatively assessed by subtracting the Phase I baseline place preference from phase III final place preference.

Quantitative measure of CPP behavior =   (Final place preference – Baseline place preference).

The experimental treatment is inferred as rewarding if a significant change towards experimental conditioned) compartment is observed. (Mathur et.al. 2011).

Darland and Dowling were the pioneers of establishing the CPP behavior in adult zebrafish (Darland & Dowling, 2001). They designed the apparatus to evaluate the addictive nature of compounds  In their model, they first recorded the baseline place preference and then confined the subjects to the least preferred compartment with exposure to cocaine for the evaluation of final place preference.

Apparatus and Equipment

The apparatus consists of a Plexiglas CPP tank approx. 52 cm (l) x 17 cm (w) x 22 cm (h) in dimensions. The tank is partitioned by two opaque dividers (one intact and the other perforated) creating a central alley for the entry of the subjects. The tank is placed inside a brown box so that the subjects cannot see the experimenter. Wide array of floor patterns can be used as visual cues, and the most realistic approach is to place sheets of flooring pattern beneath the apparatus.

The fish’s activity inside the CPP tank can be recorded with the help of Noldus Ethovision XT placed on the overhead rack at the top of the tank. Floor patterns should provide enough contrast so that the tracking of the Noldus Ethovision XT is consistent and not blocked.

Training Protocol

The purpose of the CPP paradigm is to evaluate reinforcing effects of subtances in zebrafish in a vehicle-treated control vs. disease model/intervention group, by assessing their baseline and final place preference behavior.

Pretreatment

The water temperature is maintained at approximately 28 °C, and the subjects are kept in 10-14 hour light/dark cycle. The subjects are group housed in tanks with recirculating distilled water and are transferred to individual tanks few days before the test. The testing room should be soundproof.

The room should be properly illuminated so that the subjects could distinguish the pattern and the tracking is consistent. White noise should be provided by a fan that is continued for the entire course of the test. Experimental tables should be covered with foam boards that serve as shock absorbers; diminishing the impact of vibrations.

Evaluation of Phase I & Phase II

The testing procedure lasts for two days. During Day 1, the baseline preference (Phase I) and the first conditioning (Phase II) are assessed.

At the start of testing, the subjects should be gently introduced to the central alley with the aid of the fishnet and allowed to explore. After 2 minutes, the dividers are carefully removed, and the subjects are permitted to explore all the compartments of the tank for 5 minutes. The base line place preference for the white and dotted pattern is recorded.

After Phase I, the subjects that depicted the place preference for the white compartment are divided into two groups, one receiving the vehicle treatment and the other receiving the treatment. The same procedure is applied to the subjects that preferred dotted environment.

Four CPP tanks are installed side by side on the experimental table with four Noldus Ethovision XT placed on overhead racks to monitor the activity inside the tank. Two tanks are filled with system water while the other two are filled with system water having an appropriate concentration of the compound under consideration.

Place the subjects into the least preferred compartment with the exposure to the substance and the subjects are permitted to swim freely for 20 minutes. After 20 minutes, the subjects are placed in transit tanks to wash off the substance externally. Now place the subjects in the preferred compartment with system water for 20 minutes. Vehicle-treated subjects are placed in both preferred and least preferred compartments.

Evaluation of Phase III

On Day 2, approx.16 hours (minimal intervening period for compound clearance) after Phase II, the subject’s final preference place for the white or dotted environment should be assessed.

Modifications

The original version of the CPP tank for zebrafish was developed by Darland and Dowling. Their apparatus consisted of a tank divided into two distinct compartments having visual cues (white versus dotted pattern). Numerous modifications have been made to the CPP paradigm with slight changes in the floor and tank patterns.

Ninkovic et.al. studied the reinforcing effects of substances with the aid of a CPP tank. Their apparatus consisted of two chambers having two distinct compartments colored brown and white. The floor of the tank had two black circles.

Kily et.al investigated the effects of various substances with CPP task. They modified the apparatus by dividing the tank into two chambers, one side having black spots and the other side having alternate black and white stripes.

Mathur et.al utilized the CPP model to study the effects of several compounds In their model, the tank was divided into white and dotted halves by approx. 8 cm wide central alley. The dotted compartment consisted of 16 blue dots approx. 2 cm in diameter in a uniform pattern.

Sample Data

The sample data represents the co-relation between ethanol concentration and change in preference (%) in zebrafish. It is evident from the graph that with the increasing ethanol conc. in the CPP tank, the subjects spend more time in the conditioned compartment.

Strengths & Limitations

Strengths

The CPP task is an ideal model for screening; identifying substances with reinforcing or aversive characteristics. It also helps to isolate mutants with altered responses.

A large number of compounds can be effectively evaluated in a reasonable time span making CPP the most likely choice for initial screening of rewarding compounds.

Limitations

Besides the advantages, finding the exact concentration of a substance can be time-consuming because of the ultra-narrow therapeutic window of substances in zebrafish. (Bevins, R, 2005)

The design of CPP tank is critical, and the experimenter should ensure that the subjects are not biased toward any compartment.

Summary and Key Points

• CPP is a widely used paradigm to evaluate the rewarding effects of psychoactive substances.
• The apparatus consists of a CPP tank, and a wide range of flooring patterns sheets can be used.
• Baseline place preference and final place preference is evaluated with the task.
• Numerous modifications have been made to the CPP paradigm with slight changes in the floor and tank patterns.